Using an immunocytochemical approach, an extensive system of neurons containing alpha-melanocyte stimulating hormone (alpha-MSH) was identified and mapped in the rat brain. Alpha-MSH throughout the rat brain was determined by combining microdissection techniques with a radioimmunoassay. The immunoreactive compound in brain was characterized by high pressure liquid chromatography combined with radioimmunoassay. The major immunoreactive component has an identical retention time a standard alpha-MSH, suggesting chemical identity of the peptides. Lesion of the arcuate nucleus, the site of alpha-MSH containing perikarya, depleted alpha-MSH throughout the brain. Alpha-MSH in the pineal gland and several brain regions investigated demonstrated a marked circadian rhythm. Alpha-MSH has also been identified in human post mortem specimens with a regional distribution similar to that of rat and cat brain. Alpha-MSH immunoreactive has also been identified in rat and human cerebrospinal fluid (CSF). Results suggest that alpha-MSH concentrations in rat CSF may provide an index for measuring alpha-MSH release in vivo. A calcium dependent in vitro alpha-MSH release has been demonstrated using slices of fresh rat hypothalami.